The microcirculation in the first days of ICU admission in critically ill COVID-19 patients is influenced by severity of disease

The objective of this study was to investigate the relationship between sublingual microcirculatory parameters and the severity of the disease in critically ill coronavirus disease 2019 (COVID-19) patients in the initial period of Intensive Care Unit (ICU) admission in a phase of the COVID-19 pandemic where patients were being treated with anti-inflammatory medication. In total, 35 critically ill COVID-19 patients were included. Twenty-one critically ill COVID-19 patients with a Sequential Organ Failure Assessment (SOFA) score below or equal to 7 were compared to 14 critically ill COVID-19 patients with a SOFA score exceeding 7. All patients received dexamethasone and tocilizumab at ICU admission. Microcirculatory measurements were performed within the first five days of ICU admission, preferably as soon as possible after admission. An increase in diffusive capacity of the microcirculation (total vessel density, functional capillary density, capillary hematocrit) and increased perfusion of the tissues by red blood cells was found in the critically ill COVID-19 patients with a SOFA score of 7–9 compared to the critically ill COVID-19 patients with a SOFA score ≤ 7. No such effects were found in the convective component of the microcirculation. These effects occurred in the presence of administration of anti-inflammatory medication.


Clinical demographics
The demographic characteristics, comorbidities, COVID-19 clinical management and clinical outcomes of the patients can be found in Additional file 1.The median age of the COVID-19 patients was 66 years (IQR: 53-70) and most of the patients were male (71.4%).All patients had a SOFA score < 10 in the first 24 h of ICU admission.The most common comorbidities were arterial hypertension (40%), diabetes (37.1%) and obesity (48.6%) with a mean BMI value of 30.0 ± 5.3 kg/m 2 .
Thirty-three patients (94.3%) were mechanically ventilated and two patients (5.7%) received nasal high-flow therapy at the time of the microcirculation measurement.Fourteen patients were transferred (40.0%) from another hospital.All patients received dexamethasone (6 mg per day, for 10 days) and tocilizumab (600 mg intravenous, once) at ICU admission in the LUMC or at the ICU of the hospital at which patients were admitted before they were transferred to the LUMC.The median time of ICU admission to the microcirculation measurement was 1 day (IQR: 0.0-2.0).On average, COVID-19 symptoms started 11.6 ± 4.8 days before the microcirculation measurement.Twelve patients (34.3%) died in the hospital, of which eleven patients (31.4%) were in the ICU.One patient (2.9%) received VV-ECMO and nine patients (25.7%) acquired a pulmonary embolism during their ICU stay.Furthermore, thirteen patients (37.1%) received methylprednisolone during their ICU stay due to persistent respiratory deterioration and no response to prone position.

Macrocirculatory hemodynamics
All clinical, laboratory and treatment variables, at the time of the microcirculation management, can be found in Additional file 2. COVID-19 patients were considered hypoxemic, as indicated by their partial pressure of oxygen (PaO 2 ) levels (9.7 kPa, IQR: 8.8-10.6)and PaO 2 /FiO 2 (PF)-ratio (19.7 ± 5.8).PaO 2 levels and PF-ratios did not significantly differ between the low and high SOFA group.Twenty-five patients (71.4%) received noradrenalin (with a maximum dose of 0.41 mg/kg/min) and twenty-six patients (74.3%) received heparin.There were no significant clinical, laboratory and treatment differences between the low and high SOFA score group, except for the total fluid balance and the cumulative fluid balance.The high SOFA score group showed a significantly higher total fluid balance (1229.3± 797.9 mL vs. 540.7 ± 820.5 mL; P = 0.019) and a higher cumulative fluid balance (2162.3± 1455.4 mL vs. 456.9± 2463.8 mL; P = 0.026).

Discussion
The main finding of this study was that COVID-19 patients with a SOFA score > 7-9 showed higher values for the diffusive microcirculatory parameters (TVD, FCD and cHct) than patients with a SOFA score ≤ 7 in the initial period of ICU admission (median time between ICU admission and the microcirculation measurement was one day).No such effects were found in the convective component of microcirculatory tissue perfusion, i.e.RBCv.Furthermore, the new tRBCp parameter, combining the diffusive and convective component of microcirculatory tissue perfusion, was higher in the COVID-19 patients with a SOFA score > 7-9 indicating a better perfusion of the tissues by RBCs.Notably, these effects occurred in the presence of the administration of antiinflammatory medication.
To contextualize these findings, it is essential to compare our results with two other notable investigations on critically ill COVID-19 patients and the microcirculation, namely the study by Favaron et.al. 11and Abou-Arab et.al. 13.
Favaron et.al.proposed that the increase in TVC, FCD and cHct reflects a "microcirculatory compensatory response", indicating the recruitment of the microcirculation to increase oxygen capacity in response to COVID-19-induced hypoxemia 11 .These effects were present in COVID-19 patients with a SOFA score < 10 but not in patients with a SOFA score ≥ 10.However, our study population consisted of critically ill COVID-19 patients with a SOFA score ranging from 5 to 9 and demonstrated that patients with a SOFA score > 7-9 had increased diffusive microcirculation compared to patients with a SOFA score ≤ 7.So from both studies, we still cannot make any statements as to the precise relation between the microcirculatory response and the severity of illness.
Abou-Arab et al. categorized patients into a severe group (i.e.having a respiratory rate ≥ 30/min of oxygen saturation of ≤ 90% on room air or signs of severe distress syndrome) and a critical group (i.e.having respiratory failure requiring mechanical ventilation of shock or organ failure that required ICU care) 13 .Their critical group, with a median SOFA score of 10 (IQR: 7-13), showed higher RBCv levels and higher vessel density, suggesting a "microcirculatory compensatory mechanism" compared to the severe group, with a median SOFA score of 3 (IQR: 1-4).In contrast, our study involved patients with a SOFA score < 10 (7 days, IQR: 6-8) and a significant proportion of our patients were mechanically ventilated during the microcirculation measurements (94.3%), presenting a distinctive clinical context.Our results differ from Abou-Arab et.al.emphasizing the complexity of microcirculatory responses at different severity levels of COVID-19.
Combining our results and those of Favaron et.al.and Abou-Arab et.al.makes it challenging to precisely explain the behavior of the diffusive aspects of the microcirculation in COVID-19 patients.The pattern evolving from Favaron's findings suggests an increase in the diffusive components in patients with a SOFA score < 10, which is lost in patients with a SOFA score ≥ 10.Although our study did not directly compare to healthy volunteers and did not include patients with a SOFA score ≥ 10, we observed an increase in diffusive parameters in patients with SOFA score > 7-9, which is still below the higher SOFA score group of Favaron et.al.(SOFA score ≥ 10), compared to patients with a SOFA score ≤ 7.As the SOFA ranges of the study populations in the Favaron and our study were different, it is thus far not possible to conclude or even speculate about patterns in the diffusive adaptation of the microcirculation.
Besides the variation in the definition of critical illness, the timing of the microcirculation assessments represents a second factor contributing to the divergent outcomes.Abou-Arab et.al.conducted measurements within 24 hours after ICU admission, capturing the early disease state of COVID-19 patients 13 .This aligns with our study, as the median time of measurement after ICU admission in our study population was 1 day (IQR: 0-2).In contrast, Favaron et.al.measured the microcirculation at a later time point, with a median of 7 days (IQR: 3-12) after ICU admission 11 , potentially providing insight into the evolving nature of microcirculatory changes over time.www.nature.com/scientificreports/ The third factor contributing to the divergent results is the choice of analysis method.Abou-Arab et.al.utilized AVA (Automated Vascular Analysis 3.2, Microvision Medical, Amsterdam, the Netherlands), a semi-automated software with manual vessel segmentation, while Favaron et.al.and our study applied Microtools, an automated full-frame analysis software.However, our approach placed a specific emphasis on the diffusive and convective components of the microcirculation, incorporating the newly introduced tRBCp parameter.Methodological distinctions in quantifying microcirculatory parameters across the studies could also have contributed to variations in the observed outcomes, emphasizing the significance of standardized analysis methods.
The observed increase in diffusive parameters (TVD, FCD and cHct) and tRBCp levels in the patients with a SOFA score > 7-9 (compared to patients with a SOFA score ≤ 7), despite no differences in respiratory parameters, like PF-ratios and PaO 2 levels between the two SOFA groups (Additional file 2), prompts an evaluation of the interpretation of microcirculatory recruitment in the sublingual area.This becomes especially relevant given that a substantial proportion of the SOFA increase in this population is linked to the respiratory component.The whole study population had a homogeneously low PF-ratio (19.7 ± 5.8) and low PaO 2 levels (9.7 kPa, IQR:8.8-10.6).We, therefore, could not link the microcirculatory differences in the two SOFA groups to the standard respiratory parameters as measured in the two SOFA groups.The concept of a "microcirculatory compensatory response" proposed by Favaron et.al. 11, although occurring in a hypoxic context, may be linked to the mechanism of microcirculatory adaptation observed in healthy mountaineers exposed to hypoxia at high altitudes 21 .The term "happy hypoxia" describes the remarkable ability of COVID-19 patients to cope with low PaO 2 levels similar to quite low PaO 2 levels at high altitudes 22 .COVID-19 patients may tolerate such low PaO 2 levels through a "microcirculatory compensatory response", achieved by reducing the diffusion distances between the capillaries (increased TVD and FCD) and shifting RBCs from the systemic circulation to the microcirculation (increased cHct) to increase their oxygen extraction capacity.Our results, however, could not confirm this.
We searched for a pathophysiologic explanation for the increased diffusive microcirculatory parameters in the patients with a SOFA score > 7-9 compared to those with a SOFA score ≤ 7.This was challenging because we also observed that these patients had a higher positive fluid balance.Therefore, one would expect lower values for the diffusive parameters due to tissue edema, leading to increased distances on the microcirculatory level [23][24][25] .Upon examining the available evidence to elucidate this phenomenon, our conclusion is limited to the speculation that in our study population hypoxia appears to be the primary stimulus.However, the interrelationship between various stimuli influencing the diffusive capacity of the microcirculation seems to be very complex.With the existing evidence, we are unable to provide a more definitive explanation.
Much more research is needed to clarify the mechanisms of microcirculatory response to hypoxia in different subpopulations, e.g.healthy mountaineers and COVID-19 patients.While our study showed differences in diffusive microcirculatory parameters in two groups with different SOFA scores, an important factor contributing to the generalizability should be emphasized and this is the fact that all patients received tocilizumab and dexamethasone which will have influenced capillary leakage due to the effect of the inflammatory medication on the vascular barrier function.Thus our results can only be extrapolated to a specific subset of critically ill patients, namely with a SOFA score ≤ 9 and all having received dexamethasone and tocilizumab.Furthermore, the difference in cHct cannot be adequately explained, as all patients in our cohort were treated with tocilizumab and dexamethasone.This suggests that further research is necessary to better understand the underlying mechanisms.It is possible that the use of these medications may influence endothelial function, and additional studies are needed to investigate the specific impact of these treatments on the vascular system.
Firstly, it is crucial to emphasize that the absence of a control group makes it challenging to attribute microcirculatory changes to hypoxia.An appropriate control group could have been a group of healthy volunteers or patients with mild or severe ARDS without a COVID-19 infection.Moreover, the absence of a power calculation or sample size estimation raises concerns about the potentially small size of the study population.However, there was almost no knowledge available regarding the microcirculatory response to COVID-19 at the time of our study, on which we could have based a formal sample size calculation.Moreover, during the overwhelming pandemic, we were focused on acquiring scientific knowledge as quickly as possible, to be able to understand COVID-19 better and be able to better treat our COVID-19 patients.Another limitation is the heterogeneity in the timing of measurements, capturing COVID-19 patients at various stages of their disease.Despite the median from ICU admission to microcirculation measurement being relatively short at just 1 day (IQR: 0-2), challenges in logistics, transfers between hospitals, and the overwhelming presence of COVID-19 patients made it difficult to perform time-standardized measurements.Furthermore, this study did not specifically focus on leukocytes and microcirculatory RBC aggregates, which could provide more insights into COVID-19-induced hyperinflammation and hypercoagulation.Lastly, it must be kept in mind that sublingual microcirculatory alterations do not reflect lung microcirculation.The lung microcirculation could have been more compromised than the sublingual microcirculation resulting in a reduced oxygen-extraction capacity of the lung causing hypoxemia.

Conclusion
In our study of a COVID-19 population receiving anti-inflammatory therapy, we found an increase in diffusive capacity (TVD, FCD and cHct) of the microcirculation and an increase in tRBCp in the patient with a SOFA score > 7-9 compared to patients with a SOFA score ≤ 7. Despite both groups displaying comparable systemic hypoxemia, as indicated by similar PaO 2 and FiO 2 levels, the observed increase in TVD and FCD raises questions about the specific microcirculatory response to COVID-19-induced hypoxemia.Comparisons with other studies highlighted the complexity of the microcirculatory response in COVID-19, with divergent outcomes linked to variations in critical illness definitions, timing of microcirculation measurements, and methodological discrepancies.The observed increase in diffusive parameters, even in patients with SOFA scores below the higher range in other studies, emphasized the nuanced nature of microcirculatory responses in COVID-19.While this increase

Inclusion of patients
Patients with a confirmed COVID-19 infection, based on a positive polymerase chain reaction (PCR) test result in combination with the presence of typical radiological findings according to COVID-19 Reporting and Data System (CO-RADS), who were admitted to the ICU of the LUMC from March 2021 to June 2021 were included in this study.Patients could be admitted to the ICU via the emergency department, the hospital ward or as a transfer from another hospital from the emergency room of the ICU.Exclusion criteria were age < 18 years and having maxillofacial trauma or known tumor(s) in the mouth or throat area.All patients were treated according to prevailing COVID-19 treatment modalities: dexamethasone (6 mg per day, for 10 days) and tocilizumab (600 mg intravenous, < 24 h after ICU admission, once) at ICU admission in the LUMC (or at the ICU of the hospital at which patients were admitted before they were transferred to the LUMC).Methylprednisolone (1000 mg per day, for 3 days) was started (after 10 days of dexamethasone) by persistent respiratory deterioration and no response to prone position to mitigate the cytokine storm and inflammatory response.
In this phase of the pandemic, every ICU patient received a high prophylactic dose of heparin.This prophylactic dose was doubled compared to the regular prophylactic dose but was still classified as prophylactic heparin.In cases where pulmonary embolism was confirmed, patients were administered therapeutic intravenous heparin.Fluid and vasopressor therapy was delivered according to standard clinical practice for clinically ill patients.

Data collection
For each patient, demographic data from the hospital's electronic patient dossier (EPD) system was collected at ICU admission.Recorded data included age, sex, body mass index (BMI), Acute Physiology and Chronic Health Evaluation (APACHE) IV score, SOFA score, being transferred from another hospital, time between the start of COVID-19 symptoms and microcirculation measurement and time between ICU admission and microcirculation measurement.Clinical outcome data were extracted from the EPD system in the three-month follow-up time and include duration of mechanical ventilation, pulmonary embolism during ICU stay, Venovenous Extracorporeal Membrane Oxygenation (VV-ECMO) during ICU stay, ICU length of stay, hospital length of stay, ICU mortality, hospital mortality and transfer to another hospital.At the time of the microcirculation measurement, hemodynamic, respiratory, laboratory and treatment data were collected.The total fluid balance, i.e. the daily balance of input and output at the time of the microcirculation measurement, and the cumulative fluid balance, i.e. the sum of the total fluid accumulation at the time of the microcirculation measurement, were also collected.

Measurements of the microcirculation
Sublingual microcirculation measurements were performed using IDF imaging (Cytocam™, Braedius Medical, Huizen, The Netherlands).The Cytocam is a third-generation HVM that enables non-invasive real-time visualization of the sublingual microcirculation.Detailed information on the working mechanism of the IDF imaging technique can be found elsewhere 8 .
The microcirculation measurement was performed within the first five days of ICU admission, preferably as soon as possible after ICU admission.First, the sublingual area was carefully cleaned with a suction or a gauze swab.The probe of the HVM was covered with a non-sterile disposable cap perpendicular to the area of interest in the sublingual area.Then, three videos of at least 3-5 s were recorded from different sublingual areas to minimize heterogeneity in the microscopic field of view according to the International guidelines 27 .Image sequences that did not meet the image quality criteria, focusing on illumination, image duration, focus, vessel content, stability and the absence of pressure induced by the probe 27,28 , were excluded from further analysis.

Analysis of the microcirculation
Analysis of the videos was performed with the MicroTools automatic software (Active Medical, Leiden, The Netherlands) on the full frame image sequences 10 .The following microcirculatory parameters were retrieved from this off-line analysis: total vessel density (TVD), functional capillary density (FCD), proportion of perfused vessels (PPV), red blood cell velocity (RBCv), cHct and tissue red blood cell perfusion (tRBCp) 29 .Only small microvessels (diameter < 20 µm) were considered for the calculation of these parameters.The microcirculatory parameters measure the convective and diffusive capacity of the microcirculation for oxygen transport for the tissues.Convection is quantified by flow parameters, such as the velocity of the red blood cells (RBCs) inside the capillaries of the microcirculation 27,29 .The diffusive capacity of the microcirculation is quantified by parameters, such as TVD, FCD and cHct 27,29 .The recently introduced tRBCp parameter defines the concept of tissue perfusion in relation to adequate oxygen availability 27,29,30 .This parameter combines the diffusive and convective components of tissue perfusion.A detailed description of the microcirculatory parameters can be found in Additional file 3.

Statistics
The Shapiro-Wilk normality test was used to determine whether the data were normally distributed.Normally distributed data were presented as means with standard deviations and non-normally distributed data were presented as medians with interquartile range (IQR).Driven by the signal provided by Favaron et.al. 11which was that the microcirculation behaved differently in the more severe (SOFA ≥ 10) versus less severe (SOFA < 10) critically ill patients, we wanted to have a scientifically reasonable contrast in our population.In our population the maximum SOFA score, unlike the study by Favaron et.al.was 9, so we established subgroups using the median SOFA score at admission 31 .A median SOFA score of 7 was subsequently selected as the threshold to categorize patients into two groups: those with low SOFA scores (SOFA ≤ 7) and those with high SOFA scores (SOFA > 7-9).Differences between the SOFA-score groups were tested with the independent sample t-test for normally distributed continuous data and with the Mann-Whitney U test for non-normally distributed continuous data.Categorical data were analyzed using the Chi-squared test or Fisher's exact test.A P-value of < 0.05 was considered statistically significant.

Figure 2 .
Figure 2. The critically ill coronavirus disease 2019 (COVID-19) patients in the high Sequential Organ Failure Assessment (SOFA) score group had a higher tissue vessel density (TVD) (A), a higher functional capillary density (FCD) (B), a higher capillary hematorcit (cHct) (C) and higher tissue red blood cell perfusion (tRBCp) (D) compared to the patients in the low SOFA score group.
Statistical analysis was performed using IBM SPSS software version 25.0 for Windows (IBM Corp. Released 2017.IBM SPSS Statistics for Windows, Version 25.0.Armonk, NY: IBM Corp).